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The temporal and spatial expression pattern of Myosin Va, Vb and VI in the mouse ovary
Gene Expression Patterns 6 (2006) 900–907 www.elsevier.com/locate/modgep
The temporal and spatial expression pattern of Myosin Va, Vb and VI in the mouse ovary Leeanne McGurk a, George Tzolovsky b, Norah Spears c, Mary Bownes
d,*
a MRC Human Genetics Unit, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, United Kingdom Department of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, United Kingdom d ICB, University of Edinburgh, Darwin Building, Kings Buildings, Edinburgh, EH9 3JR, United Kingdom b
c
Received 9 January 2006; received in revised form 23 February 2006; accepted 2 March 2006 Available online 6 March 2006
Abstract There are 16 classes of unconventional myosins. Class V myosins have been shown to be involved in transporting cargo to and from the cell periphery. Class VI myosins have also been shown to transport cargo from the cell periphery, although it seems that these proteins have many roles which include the mediation of cell migration and stereocillia stabilisation. With the requirement of myosin VI for Drosophila oogenesis, the localised expression of Myosin V in the developing egg chamber and recent mounting evidence which links myosin VI to the migration of human ovarian cancer cell lines, we wanted to investigate the expression pattern of these two myosin classes in the normal mouse ovary. Here we show that these myosins are expressed, localised and regulated within the oocyte and granulosa cells of the developing mouse follicle. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Class V and VI unconventional myosin; Mouse ovarian follicle development; RNA localisation; Immunohistochemistry; Genomic organisation
1. Results and discussion All Myosins are characterised by a mechanochemical head domain, a neck domain, an a-helical coiled-coil domain and a globular tail domain (Mermall et al., 1998; Mooseker and Cheney, 1995). Binding of the head to actin results in the hydrolysis of ATP and a conformational change that is recognised by the neck domain. The neck, in turn, acts as a lever creating the power stroke required for movement along the actin filament (Vale and Milligan, 2000). The neck contains IQ domains; binding sites for the calcium binding protein calmodulin, which when bound to the neck domain of Myosin V, is thought to mediate the *
Corresponding author. Tel.: +44 0131 650 5369; fax: +44 0131 650 5371. E-mail addresses: [email protected] (L. McGurk), [email protected] (G. Tzolovsky), [email protected] (N. Spears), [email protected] (M. Bownes). 1567-133X/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2006.03.002
conformation required for the power stroke and processive movement (Bahloul et al., 2004; Krementsov et al., 2004). The coiled-coil domain of the tail is thought to hold the dimer together, leaving the globular tail to associate with cargo such as organelles and mRNA (Long et al., 1997; Mercer et al., 1991; Mermall et al., 1994; Mermall and Miller, 1995). The role of Myosin Va, Vb and VI in mammals has been widely researched and has provided insight into the cellular roles these proteins play. Myosin Va has been shown to be involved in secretion (Mercer et al., 1991; Wu et al., 1997) whereas Myosin Vb has been shown to be involved in plasma membrane recycling in non-polarised and polarised cells (Casanova et al., 1999; Green et al., 1997; Ren et al., 1998; Ullrich et al., 1996). In the inner ear Myosin VI localises to the base of the stereocilia, a region of endocytosis (Hasson, 1997; Hasson et al., 1997; Hasson and Mooseker, 1994; Self et al., 1999). In some cell types Myosin VI co-localises to clathrin coated pits via an insert in the tail
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domain (Buss et al., 2001; Morris et al., 2002). It is thought that this role in early endocytosis may promote the anchorage of plasma membrane invaginations such as stereocillia and microvilli (Altman et al., 2004). As well as functioning in the early endocytotic pathway Myosin VI is required for secretion from the trans-Golgi network and is essential for maintaining the Golgi structure (Warner et al., 2003). Myosin VI may also promote the anchorage of invaginations by mediating the assembly of actin filaments, as has been shown in the fly testes during sperm individualisation (Rogat and Miller, 2002). Myosin VI also localises to the leading edge of fibroblasts, where it may mediate cell migration (Buss et al., 1998). Recently Myosin VI was found to promote the migration of human and mouse ovarian cancer cells (Yoshida et al., 2004). Myosin V and VI have also been studied in invertebrates, including during Drosophila oogenesis. myosin VI is expressed in the migrating epithelial cells of the egg chamber (border cells, centripetal cells and the dorsal appendage follicle cells), prior to and during migration (Deng et al., 1999; Geisbrecht and Montell, 2002). More recently it has been found that the interaction of Myosin VI with cell junctions is essential for maintaining epithelial cell integrity in the egg chamber (Millo, Lazou, Hu and Bownes, unpublished). Myosin VI is also required for the transport of nurse cell material to the oocyte (Bohrmann, 1997). Myosin V is expressed in the nurse cells from the early stages of oogenesis, and is in the oocyte later in egg chamber development, where it associates with the peripheral actin cytoskeleton (Mermall et al., 2005). Mouse follicle development, like Drosophila oogenesis, involves complex cell–cell interactions between a germ cell and somatic cells. At birth the mouse oocyte is arrested in meiotic prophase I and is contained within a resting primordial follicle (Albertini et al., 2001). At any one time, the majority of primordial follicles will remain in this quiescent state, with a small proportion of follicles leaving the resting pool to undergo growth initiation. The granulosa cells of the developing primary follicle become cuboidal and divide to form concentric layers. As the pre-antral follicle continues to grow a second somatic layer, called the thecal layer, forms around the granulosa cells. Follicular secretions build up within the granulosa cell layers to form the antral cavity (Bostrom and Odeblad, 1952; Zachariae, 1957). The antral cavity increases in size and separates the oocyte with the associating cumulus granulosa cells from the thecal layer and the associating mural granulosa cells to form the cumulus–oocyte complex, COC. The mural granulosa cells, thecal cells and the outer surface epithelium, undergo a series of morphological changes (Byskov, 1969; Martin and Talbot, 1987; Talbot et al., 1987) and eventually the apical follicle and the COC is released into the oviduct (Martin and Talbot, 1987). In this paper, we investigate the expression of Myosin Va, Vb and V1 in RNA and protein during follicle development in the mouse.
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1.1. Temporal expression of myosin Va, Vb and VI RNA All three transcripts are detected in the immature ovary (3 weeks) and mature ovary (6 weeks) using RT-PCR. PMSG, pregnant mare serum gonadotrophin, is injected into female mice in order to promote the development of late antral follicles. Injection of hCG, human chronic gonadotrophin, 48 h later then induces ovulation. myosin Va, Vb and VI are expressed in ovaries just before ovulation (PMSG treated) and just after ovulation (PMSG and hCG treated) (Fig. 1). Granulosa cells and oocytes were dissected from late antral follicles and RT-PCR was used to detect the expression of these three transcripts. All three RNAs are expressed in the granulosa cells, but only myosin Vb is detected in the oocyte of late antral follicles (Fig. 1).
Fig. 1. Temporal expression of myosin Va, Vb and VI in the mouse ovary. (a) The expression levels of myosin Va, Vb and VI were compared in the ovaries of an immature mouse (3 week) and of a mature mouse (6 week). The mouse brain was used as a positive control and b-actin was used as the internal control for all RT-PCRs. (b) Expression of myosin Va, Vb and VI was detected in the ovary prior to ovulation i.e., from PMSG treated ovaries, and after ovulation i.e., from PMSG and hCG treated ovaries. (c) myosin Va was detected at low levels in mouse oocytes and at higher levels in the granulosa cells. myosin Vb was detected in both oocytes and granulosa, whereas myosin VI was only be detected in the granulosa cells.
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1.2. Spatial expression of myosin Va, Vb and VI RNA Cell specific expression of myosin Va, Vb and VI is detected in the adult mouse ovary. Low levels of myosin Va, Vb and VI are seen in the stromal tissue and in the granulosa cells of follicles of all stages. myosin Va and VI localise to the oocyte periphery in early follicles and antral follicles (Figs. 2a and c). myosin Vb is seen as punctate staining throughout the oocyte of early and antral follicles (Fig. 2b). The in situ data suggests that all three transcripts are detected in the oocyte from early follicles through to antral follicles. None of the follicles shown following in situ hybridisation are late antral. The RT-PCR suggests that only the myosin Vb transcript remains in the oocyte of
late antral follicles. The RT-PCR also confirms that the three transcripts are expressed in the granulosa cells. Thus although all three mRNAs are present during mouse follicle their expression patterns and localisation in the oocyte varied. We therefore investigated the distribution of these proteins as a step towards investigating their function in oogenesis. 1.3. Spatial expression of Myosin Va, Vb and VI protein in the developing mouse follicle Antibodies directed to peptides within the proteins were obtained commercially: all were checked for detection of full length protein by immunoblotting (Fig. 3). The
Fig. 2. myosin Va, Vb and VI mRNA localisation in the mouse follicle. (a) myosin Va RNA localises to the periphery of the oocyte and in the granulosa cells of growing pre-antral and antral follicles. (b) myosin Vb RNA localises throughout the ooplasm of pre-antral and antral follicles. (c) myosin VI RNA localises to the periphery of the oocyte of growing follicles. O, oocyte; GC, granulosa cells; T, thecal cells and AC, antral cavity.
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Fig. 3. Detection of myosin Va, Vb and VI protein by Western blot. (a) Full length Myosin Va epitope in the mouse brain is detected at 188 kDa along with two smaller, possibly degraded products. (b) Full length Myosin Vb in the mouse brain is detected at 214 kDa, two extra bands were also seen at around 150 kDa. (c) Full length Myosin VI is detected at 150 kDa in both the brain and ovary of a 6-week-old mouse.
antibodies were subsequently used to observe protein localisation in the mouse ovary. All proteins are cytoplasmic and all have an expression pattern which changes throughout follicle development. In some early follicles Myosin Va concentrates at the oocyte periphery (Fig. 4c, arrows). In growing pre-antral follicles (Fig. 4d) very strong punctuate staining (asterisk) and low level diffuse staining can be seen in the ooplasm and in the cytoplasm of the granulosa cells, however no staining is seen at the granulosa cell membrane next to the oocyte (arrow). Myosin Va remains high in the oocytes of antral follicles, again there is punctuate localisation and diffuse staining throughout the ooplasm (Fig. 4e). The difference between cumulus granulosa cells and mural granulosa cells of the antral follicle is distinct (Fig. 4e). Strong staining is seen at the lateral membranes between cumulus granulosa cells but not at the membrane adjacent to the oocyte. The staining is very strong between some mural and cumulus cells, possibly at points of antral cavity formation, and is also seen at the antral edge of cumulus and mural granulosa cells. Staining between adjacent mural granulosa cells is less prominent and virtually no staining is seen between the basal membrane and mural granulosa cells or in the thecal layer (arrow). In ovaries treated with PMSG there is much less punctate staining of the oocyte than in the non-PMSG treated ovary. Myosin Va staining between adjacent cumulus cells decreases whereas Myosin Va in the mural granulosa cells
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increases (Fig. 4e). Staining exists at the membranes between adjacent mural granulosa cells, between the mural granulosa cells and the thecal cells and staining is also seen in the thecal layer. Myosin Vb localisation was not as strong as Myosin Va. Growing pre-antral follicles have staining in the granulosa cells around the oocyte (Fig. 5a, arrows). In antral follicles staining is higher in the granulosa cells but less is seen in the cells adjacent to the basement membrane (Fig. 5b, arrow). Staining is strongest at the plasma membranes between granulosa cells. Myosin VI is present in all stages of follicles. At very early stages of follicle development there is a distinct ring around the oocyte (Fig. 6c, arrows). By the primary stage (Fig. 6d) Myosin VI staining is very strong at the oocyte periphery and at the edge of the adjacent granulosa cells some staining is also seen between the granulosa cells and the basal lamina (arrow). In the early antral follicle (Fig. 6e) Myosin VI staining is seen at the oocyte edge of some of the granulosa cells, between cumulus granulosa cells, at the antral edge of the cumulus granulosa cells and the mural granulosa cells. Mural granulosa cells near the basement membrane were not stained and no localisation is seen in the thecal cell layers (Fig. 6e and f). In Summary myosin Va RNA and protein is expressed and localised to specific regions within the oocytes of developing follicles, the RNA is found at the periphery whereas the protein localises to distinct regions within the ooplasm. The protein is present in the granulosa cells and is more abundant in the cumulus granulosa cells than the mural granulosa cells and is absent in the thecal cells and the adjacent granulosa cell membranes. After PMSG treatment the expression is higher in the mural granulosa cells and the thecal cells. myosin Vb RNA is present throughout the ooplasm of follicles at all stages. The protein, however, is only detected at the very edge of the oocyte of some early follicles. The RNA is detected at low levels in the granulosa cells, as is the protein, which is more abundant in the cumulus granulosa cells than the mural granulosa cells. myosin VI RNA also localises to the edge of the oocyte in follicles of all stages. The protein, however, is only detected at the oocyte periphery of early follicles, after which the protein is only seen in the granulosa cells. Again the staining is stronger in the cumulus granulosa cells than the mural granulosa cells and no staining is seen in the mural granulosa cells adjacent to the theca or in the theca of antral follicles. 2. Materials and methods 2.1. Mouse husbandry and hormone treatments The F1 mice were collected from a CBA/C57BLJ6 cross and maintained for up to 6 weeks. 3 week old mice were injected with 5IU of PMSG. 48 hours later the same mice were injected with 5IU of hCG. The following morning the ovaries were collected and either fixed in 4% paraformaldehyde or frozen in liquid nitrogen. Control ovaries were
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Fig. 4. Myosin Va protein in the mouse ovary. (a) Myosin Va localises to oocytes and granulosa cells in a 3-week-old ovary. (b) The 3 week old ovary incubated without the primary antibody. (c) Myosin Va localising to the edge of the oocyte in two early follicles (arrows). (d) In the pre-antral follicle, Myosin Va localises to distinct points within the oocyte and is also seen in the cytoplasm of the granulosa cells with no staining seen on the membrane adjacent to the oocyte (arrow). (e) In the antral follicle staining is still strong in the oocyte and in the cytoplasm of the granulosa cells, no staining was seen in the thecal layer or adjacent mural granulosa cells. (f) In the large antral follicle Myosin Va decreases in the oocyte and decreases in the cumulus granulosa cells. Levels are now higher in the mural granulsoa cells, thecal cells and in the outer surface epithelium. O, oocyte; GC, granulosa cells; T, thecal cells; AC, antral cavity and OSE, outer surface epithelium. Scale bars: a–b = 100 lm, c = 20 lm and d–f = 40 lm.
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Fig. 5. Myosin Vb protein in the mouse ovary. (a) Myosin Vb can be seen in the oocyte and surrounding granulosa cells of the primary follicle and staining can be seen at the membrane adjacent to the oocyte (arrows). (b) Myosin Vb localises more strongly to the lateral plasma membranes of the cumulus granulosa cells compared to the mural granulosa cells. No staining is seen in the theca or adjacent granulosa cells (arrow). (c) Without Myosin Vb antibody. O, oocyte; GC, granulosa cells; T, thecal cells; AC, antral cavity and OSE, outer surface epithelium. All scale bars = 40 lm. collected 24 h after PMSG injection. Tissue was either snap frozen for use in RT-PCR or processed and embedded in wax, following normal histological procedures, for use in RNA in situ and immunohistochemistry.
2.2. RNA isolation and RT-PCR
T3 and T7 polymerases from Amersham and with the DIG RNA labelling mix from Roche. Sections were prepared and dehydrated using a standard histology protocol. After washes in PBS the tissue was digested with proteinase K (12 lg/ml final concentration) at 25 °C, the reaction was stopped with 2 mg/ml glycine. Tissue was equilibriated in PBS/PHS and pre-hybridised at 61–65 °C. The probe was diluted 250–500 times in pre-hybridisation solution and incubated overnight at 61 °C. The sections were washed and incubated in 25% formamide at 60 °C. The probe was detected with the anti-DIG/HRP antibody (DakoCytomation P5104) and washed. The slides were incubated in TSA reagent, 1 in 50 in amplification buffer (Perkin-Elmer) and were washed before being mounted.
RNA was prepared from four 3-week-old mouse ovaries and a small slice of mouse brain (0.5 cm2) in TrizolÓ. Reverse transcription was carried out using Invitrogen’s Superscript II and reactions followed the manufacturer’s instructions. 0.5 lg of cDNA was added to a reaction containing Qiagen Taq according to the manufacturer’s instructions. Late preantral follicles were dissected from ovaries and RNA was then isolated from both granulosa cells and oocytes. The granulosa cells can be considered a clean population of cells. It is, however, difficult to ensure that oocytes are entirely free from granulosa cells; as such the mRNA obtained from oocytes could be contaminated by a very small amount of granulosa cells. b-Actin was the internal control. The primers used were; mMyoVa FP1: AGC TCA ACT CCT TCC ACT C, mMyoVa RP2: ACA CAC CCC TTT ATC CTT CC, mMyoVb FP1: ACC CCT CCA TTT CCA CTT CC, mMyoVb RP2: GCT GCT TCT TCA GCT TCC TC, MMyoVbFP14: GTC ATG ACG TAC AGC GAG CTC, MMyoVbFP4: GCC AAG CAC ATC TAT GCT CA, mMyo VI F: ATG GAA GAT GGA AAG CCC G, mMyo VI R: GCT CCT CTT TGC CTT GAA C, b-Actin A: TAC CTC ATG AAG ATC CTG ACC GAG, b-Actin B: CTC CTG CTT GCT GAT CCA CAT CTG.
Antibodies directed to peptides within the proteins were obtained commercially (Myosin Va: Sigma, M4812, Myosin Vb: BD Transduction Laboratories, M11220, Myosin VI: Sigma, M5187). Protein was extracted from 1 g of dissected tissue in RIPA buffer containing the appropriate concentration of Roche’s protease inhibitor cocktail tablets. Fifty micrograms of heat denatured cell lysate was loaded onto either a 10% polyacrylamide separating mix and a 5% polyacrylamide stacking gel (when using the semi-dry transfer method) or onto Invitrogen’s 4-12% Bis-Tris gel (when using the wet transfer method).
2.3. Probe preparation and RNA in situ
2.5. Immunohistochemistry
Probes were transcribed from 5034 to 5893 bp of myosin Va (NM_010864), 3491–4331 bp of myosin Vb (NM_201600) and 3345– 4238 bp of myosin VI (NMU49739). Transcription was carried out using
Sections were prepared and dehydrated following normal histology procedure. Heat induced antigen retrieval was carried out in citrate buffer pH 6 when detecting Myosin Vb and VI. Antibodies were used at pre-de-
2.4. Immunoblot detection
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Fig. 6. Myosin VI protein in the developing mouse follicle. (a) Myosin VI localises strongly to the cytoplasm of granulosa cells. (b) Without Myosin VI antibody. (c) Myosin VI localises to oocyte periphery in all very early follicles (arrows). (d) In the growing primary follicle Myosin VI staining in the ooplasm is strong and is also in the granulosa cells (arrow). (e) No Myosin VI is seen in the oocyte of antral follicles. Myosin VI is high at the lateral membranes of granulosa cells and no staining is seen in the lateral or basal membranes of mural granulosa cells adjacent to the theca, however staining is seen in apical membranes in contact with the granulosa cells (arrow). (f) In the large antral follicle Myosin VI is strong in the cytoplasm of mural and cumulus granulosa cells but low in the thecal cells. O, oocyte; GC, granulosa cells; T, thecal cells; AC, antral cavity and OSE, outer surface epithelium. Scale bars: a–b = 100 lm, c–d = 30 lm and e–f = 30 lm.
L. McGurk et al. / Gene Expression Patterns 6 (2006) 900–907 termined concentrations. Sections incubated with the mouse Myosin Vb antibody were first blocked with the Fab fragment of mouse IgG. Fluorescent secondary antibodies were used to detect antibody localisation.
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The temporal and spatial expression pattern of Myosin Va, Vb and VI in the mouse ovary Leeanne McGurk a, George Tzolovsky b, Norah Spears c, Mary Bownes
d,*
a MRC Human Genetics Unit, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, United Kingdom Department of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, United Kingdom d ICB, University of Edinburgh, Darwin Building, Kings Buildings, Edinburgh, EH9 3JR, United Kingdom b
c
Received 9 January 2006; received in revised form 23 February 2006; accepted 2 March 2006 Available online 6 March 2006
Abstract There are 16 classes of unconventional myosins. Class V myosins have been shown to be involved in transporting cargo to and from the cell periphery. Class VI myosins have also been shown to transport cargo from the cell periphery, although it seems that these proteins have many roles which include the mediation of cell migration and stereocillia stabilisation. With the requirement of myosin VI for Drosophila oogenesis, the localised expression of Myosin V in the developing egg chamber and recent mounting evidence which links myosin VI to the migration of human ovarian cancer cell lines, we wanted to investigate the expression pattern of these two myosin classes in the normal mouse ovary. Here we show that these myosins are expressed, localised and regulated within the oocyte and granulosa cells of the developing mouse follicle. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Class V and VI unconventional myosin; Mouse ovarian follicle development; RNA localisation; Immunohistochemistry; Genomic organisation
1. Results and discussion All Myosins are characterised by a mechanochemical head domain, a neck domain, an a-helical coiled-coil domain and a globular tail domain (Mermall et al., 1998; Mooseker and Cheney, 1995). Binding of the head to actin results in the hydrolysis of ATP and a conformational change that is recognised by the neck domain. The neck, in turn, acts as a lever creating the power stroke required for movement along the actin filament (Vale and Milligan, 2000). The neck contains IQ domains; binding sites for the calcium binding protein calmodulin, which when bound to the neck domain of Myosin V, is thought to mediate the *
Corresponding author. Tel.: +44 0131 650 5369; fax: +44 0131 650 5371. E-mail addresses: [email protected] (L. McGurk), [email protected] (G. Tzolovsky), [email protected] (N. Spears), [email protected] (M. Bownes). 1567-133X/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2006.03.002
conformation required for the power stroke and processive movement (Bahloul et al., 2004; Krementsov et al., 2004). The coiled-coil domain of the tail is thought to hold the dimer together, leaving the globular tail to associate with cargo such as organelles and mRNA (Long et al., 1997; Mercer et al., 1991; Mermall et al., 1994; Mermall and Miller, 1995). The role of Myosin Va, Vb and VI in mammals has been widely researched and has provided insight into the cellular roles these proteins play. Myosin Va has been shown to be involved in secretion (Mercer et al., 1991; Wu et al., 1997) whereas Myosin Vb has been shown to be involved in plasma membrane recycling in non-polarised and polarised cells (Casanova et al., 1999; Green et al., 1997; Ren et al., 1998; Ullrich et al., 1996). In the inner ear Myosin VI localises to the base of the stereocilia, a region of endocytosis (Hasson, 1997; Hasson et al., 1997; Hasson and Mooseker, 1994; Self et al., 1999). In some cell types Myosin VI co-localises to clathrin coated pits via an insert in the tail
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domain (Buss et al., 2001; Morris et al., 2002). It is thought that this role in early endocytosis may promote the anchorage of plasma membrane invaginations such as stereocillia and microvilli (Altman et al., 2004). As well as functioning in the early endocytotic pathway Myosin VI is required for secretion from the trans-Golgi network and is essential for maintaining the Golgi structure (Warner et al., 2003). Myosin VI may also promote the anchorage of invaginations by mediating the assembly of actin filaments, as has been shown in the fly testes during sperm individualisation (Rogat and Miller, 2002). Myosin VI also localises to the leading edge of fibroblasts, where it may mediate cell migration (Buss et al., 1998). Recently Myosin VI was found to promote the migration of human and mouse ovarian cancer cells (Yoshida et al., 2004). Myosin V and VI have also been studied in invertebrates, including during Drosophila oogenesis. myosin VI is expressed in the migrating epithelial cells of the egg chamber (border cells, centripetal cells and the dorsal appendage follicle cells), prior to and during migration (Deng et al., 1999; Geisbrecht and Montell, 2002). More recently it has been found that the interaction of Myosin VI with cell junctions is essential for maintaining epithelial cell integrity in the egg chamber (Millo, Lazou, Hu and Bownes, unpublished). Myosin VI is also required for the transport of nurse cell material to the oocyte (Bohrmann, 1997). Myosin V is expressed in the nurse cells from the early stages of oogenesis, and is in the oocyte later in egg chamber development, where it associates with the peripheral actin cytoskeleton (Mermall et al., 2005). Mouse follicle development, like Drosophila oogenesis, involves complex cell–cell interactions between a germ cell and somatic cells. At birth the mouse oocyte is arrested in meiotic prophase I and is contained within a resting primordial follicle (Albertini et al., 2001). At any one time, the majority of primordial follicles will remain in this quiescent state, with a small proportion of follicles leaving the resting pool to undergo growth initiation. The granulosa cells of the developing primary follicle become cuboidal and divide to form concentric layers. As the pre-antral follicle continues to grow a second somatic layer, called the thecal layer, forms around the granulosa cells. Follicular secretions build up within the granulosa cell layers to form the antral cavity (Bostrom and Odeblad, 1952; Zachariae, 1957). The antral cavity increases in size and separates the oocyte with the associating cumulus granulosa cells from the thecal layer and the associating mural granulosa cells to form the cumulus–oocyte complex, COC. The mural granulosa cells, thecal cells and the outer surface epithelium, undergo a series of morphological changes (Byskov, 1969; Martin and Talbot, 1987; Talbot et al., 1987) and eventually the apical follicle and the COC is released into the oviduct (Martin and Talbot, 1987). In this paper, we investigate the expression of Myosin Va, Vb and V1 in RNA and protein during follicle development in the mouse.
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1.1. Temporal expression of myosin Va, Vb and VI RNA All three transcripts are detected in the immature ovary (3 weeks) and mature ovary (6 weeks) using RT-PCR. PMSG, pregnant mare serum gonadotrophin, is injected into female mice in order to promote the development of late antral follicles. Injection of hCG, human chronic gonadotrophin, 48 h later then induces ovulation. myosin Va, Vb and VI are expressed in ovaries just before ovulation (PMSG treated) and just after ovulation (PMSG and hCG treated) (Fig. 1). Granulosa cells and oocytes were dissected from late antral follicles and RT-PCR was used to detect the expression of these three transcripts. All three RNAs are expressed in the granulosa cells, but only myosin Vb is detected in the oocyte of late antral follicles (Fig. 1).
Fig. 1. Temporal expression of myosin Va, Vb and VI in the mouse ovary. (a) The expression levels of myosin Va, Vb and VI were compared in the ovaries of an immature mouse (3 week) and of a mature mouse (6 week). The mouse brain was used as a positive control and b-actin was used as the internal control for all RT-PCRs. (b) Expression of myosin Va, Vb and VI was detected in the ovary prior to ovulation i.e., from PMSG treated ovaries, and after ovulation i.e., from PMSG and hCG treated ovaries. (c) myosin Va was detected at low levels in mouse oocytes and at higher levels in the granulosa cells. myosin Vb was detected in both oocytes and granulosa, whereas myosin VI was only be detected in the granulosa cells.
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1.2. Spatial expression of myosin Va, Vb and VI RNA Cell specific expression of myosin Va, Vb and VI is detected in the adult mouse ovary. Low levels of myosin Va, Vb and VI are seen in the stromal tissue and in the granulosa cells of follicles of all stages. myosin Va and VI localise to the oocyte periphery in early follicles and antral follicles (Figs. 2a and c). myosin Vb is seen as punctate staining throughout the oocyte of early and antral follicles (Fig. 2b). The in situ data suggests that all three transcripts are detected in the oocyte from early follicles through to antral follicles. None of the follicles shown following in situ hybridisation are late antral. The RT-PCR suggests that only the myosin Vb transcript remains in the oocyte of
late antral follicles. The RT-PCR also confirms that the three transcripts are expressed in the granulosa cells. Thus although all three mRNAs are present during mouse follicle their expression patterns and localisation in the oocyte varied. We therefore investigated the distribution of these proteins as a step towards investigating their function in oogenesis. 1.3. Spatial expression of Myosin Va, Vb and VI protein in the developing mouse follicle Antibodies directed to peptides within the proteins were obtained commercially: all were checked for detection of full length protein by immunoblotting (Fig. 3). The
Fig. 2. myosin Va, Vb and VI mRNA localisation in the mouse follicle. (a) myosin Va RNA localises to the periphery of the oocyte and in the granulosa cells of growing pre-antral and antral follicles. (b) myosin Vb RNA localises throughout the ooplasm of pre-antral and antral follicles. (c) myosin VI RNA localises to the periphery of the oocyte of growing follicles. O, oocyte; GC, granulosa cells; T, thecal cells and AC, antral cavity.
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Fig. 3. Detection of myosin Va, Vb and VI protein by Western blot. (a) Full length Myosin Va epitope in the mouse brain is detected at 188 kDa along with two smaller, possibly degraded products. (b) Full length Myosin Vb in the mouse brain is detected at 214 kDa, two extra bands were also seen at around 150 kDa. (c) Full length Myosin VI is detected at 150 kDa in both the brain and ovary of a 6-week-old mouse.
antibodies were subsequently used to observe protein localisation in the mouse ovary. All proteins are cytoplasmic and all have an expression pattern which changes throughout follicle development. In some early follicles Myosin Va concentrates at the oocyte periphery (Fig. 4c, arrows). In growing pre-antral follicles (Fig. 4d) very strong punctuate staining (asterisk) and low level diffuse staining can be seen in the ooplasm and in the cytoplasm of the granulosa cells, however no staining is seen at the granulosa cell membrane next to the oocyte (arrow). Myosin Va remains high in the oocytes of antral follicles, again there is punctuate localisation and diffuse staining throughout the ooplasm (Fig. 4e). The difference between cumulus granulosa cells and mural granulosa cells of the antral follicle is distinct (Fig. 4e). Strong staining is seen at the lateral membranes between cumulus granulosa cells but not at the membrane adjacent to the oocyte. The staining is very strong between some mural and cumulus cells, possibly at points of antral cavity formation, and is also seen at the antral edge of cumulus and mural granulosa cells. Staining between adjacent mural granulosa cells is less prominent and virtually no staining is seen between the basal membrane and mural granulosa cells or in the thecal layer (arrow). In ovaries treated with PMSG there is much less punctate staining of the oocyte than in the non-PMSG treated ovary. Myosin Va staining between adjacent cumulus cells decreases whereas Myosin Va in the mural granulosa cells
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increases (Fig. 4e). Staining exists at the membranes between adjacent mural granulosa cells, between the mural granulosa cells and the thecal cells and staining is also seen in the thecal layer. Myosin Vb localisation was not as strong as Myosin Va. Growing pre-antral follicles have staining in the granulosa cells around the oocyte (Fig. 5a, arrows). In antral follicles staining is higher in the granulosa cells but less is seen in the cells adjacent to the basement membrane (Fig. 5b, arrow). Staining is strongest at the plasma membranes between granulosa cells. Myosin VI is present in all stages of follicles. At very early stages of follicle development there is a distinct ring around the oocyte (Fig. 6c, arrows). By the primary stage (Fig. 6d) Myosin VI staining is very strong at the oocyte periphery and at the edge of the adjacent granulosa cells some staining is also seen between the granulosa cells and the basal lamina (arrow). In the early antral follicle (Fig. 6e) Myosin VI staining is seen at the oocyte edge of some of the granulosa cells, between cumulus granulosa cells, at the antral edge of the cumulus granulosa cells and the mural granulosa cells. Mural granulosa cells near the basement membrane were not stained and no localisation is seen in the thecal cell layers (Fig. 6e and f). In Summary myosin Va RNA and protein is expressed and localised to specific regions within the oocytes of developing follicles, the RNA is found at the periphery whereas the protein localises to distinct regions within the ooplasm. The protein is present in the granulosa cells and is more abundant in the cumulus granulosa cells than the mural granulosa cells and is absent in the thecal cells and the adjacent granulosa cell membranes. After PMSG treatment the expression is higher in the mural granulosa cells and the thecal cells. myosin Vb RNA is present throughout the ooplasm of follicles at all stages. The protein, however, is only detected at the very edge of the oocyte of some early follicles. The RNA is detected at low levels in the granulosa cells, as is the protein, which is more abundant in the cumulus granulosa cells than the mural granulosa cells. myosin VI RNA also localises to the edge of the oocyte in follicles of all stages. The protein, however, is only detected at the oocyte periphery of early follicles, after which the protein is only seen in the granulosa cells. Again the staining is stronger in the cumulus granulosa cells than the mural granulosa cells and no staining is seen in the mural granulosa cells adjacent to the theca or in the theca of antral follicles. 2. Materials and methods 2.1. Mouse husbandry and hormone treatments The F1 mice were collected from a CBA/C57BLJ6 cross and maintained for up to 6 weeks. 3 week old mice were injected with 5IU of PMSG. 48 hours later the same mice were injected with 5IU of hCG. The following morning the ovaries were collected and either fixed in 4% paraformaldehyde or frozen in liquid nitrogen. Control ovaries were
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Fig. 4. Myosin Va protein in the mouse ovary. (a) Myosin Va localises to oocytes and granulosa cells in a 3-week-old ovary. (b) The 3 week old ovary incubated without the primary antibody. (c) Myosin Va localising to the edge of the oocyte in two early follicles (arrows). (d) In the pre-antral follicle, Myosin Va localises to distinct points within the oocyte and is also seen in the cytoplasm of the granulosa cells with no staining seen on the membrane adjacent to the oocyte (arrow). (e) In the antral follicle staining is still strong in the oocyte and in the cytoplasm of the granulosa cells, no staining was seen in the thecal layer or adjacent mural granulosa cells. (f) In the large antral follicle Myosin Va decreases in the oocyte and decreases in the cumulus granulosa cells. Levels are now higher in the mural granulsoa cells, thecal cells and in the outer surface epithelium. O, oocyte; GC, granulosa cells; T, thecal cells; AC, antral cavity and OSE, outer surface epithelium. Scale bars: a–b = 100 lm, c = 20 lm and d–f = 40 lm.
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Fig. 5. Myosin Vb protein in the mouse ovary. (a) Myosin Vb can be seen in the oocyte and surrounding granulosa cells of the primary follicle and staining can be seen at the membrane adjacent to the oocyte (arrows). (b) Myosin Vb localises more strongly to the lateral plasma membranes of the cumulus granulosa cells compared to the mural granulosa cells. No staining is seen in the theca or adjacent granulosa cells (arrow). (c) Without Myosin Vb antibody. O, oocyte; GC, granulosa cells; T, thecal cells; AC, antral cavity and OSE, outer surface epithelium. All scale bars = 40 lm. collected 24 h after PMSG injection. Tissue was either snap frozen for use in RT-PCR or processed and embedded in wax, following normal histological procedures, for use in RNA in situ and immunohistochemistry.
2.2. RNA isolation and RT-PCR
T3 and T7 polymerases from Amersham and with the DIG RNA labelling mix from Roche. Sections were prepared and dehydrated using a standard histology protocol. After washes in PBS the tissue was digested with proteinase K (12 lg/ml final concentration) at 25 °C, the reaction was stopped with 2 mg/ml glycine. Tissue was equilibriated in PBS/PHS and pre-hybridised at 61–65 °C. The probe was diluted 250–500 times in pre-hybridisation solution and incubated overnight at 61 °C. The sections were washed and incubated in 25% formamide at 60 °C. The probe was detected with the anti-DIG/HRP antibody (DakoCytomation P5104) and washed. The slides were incubated in TSA reagent, 1 in 50 in amplification buffer (Perkin-Elmer) and were washed before being mounted.
RNA was prepared from four 3-week-old mouse ovaries and a small slice of mouse brain (0.5 cm2) in TrizolÓ. Reverse transcription was carried out using Invitrogen’s Superscript II and reactions followed the manufacturer’s instructions. 0.5 lg of cDNA was added to a reaction containing Qiagen Taq according to the manufacturer’s instructions. Late preantral follicles were dissected from ovaries and RNA was then isolated from both granulosa cells and oocytes. The granulosa cells can be considered a clean population of cells. It is, however, difficult to ensure that oocytes are entirely free from granulosa cells; as such the mRNA obtained from oocytes could be contaminated by a very small amount of granulosa cells. b-Actin was the internal control. The primers used were; mMyoVa FP1: AGC TCA ACT CCT TCC ACT C, mMyoVa RP2: ACA CAC CCC TTT ATC CTT CC, mMyoVb FP1: ACC CCT CCA TTT CCA CTT CC, mMyoVb RP2: GCT GCT TCT TCA GCT TCC TC, MMyoVbFP14: GTC ATG ACG TAC AGC GAG CTC, MMyoVbFP4: GCC AAG CAC ATC TAT GCT CA, mMyo VI F: ATG GAA GAT GGA AAG CCC G, mMyo VI R: GCT CCT CTT TGC CTT GAA C, b-Actin A: TAC CTC ATG AAG ATC CTG ACC GAG, b-Actin B: CTC CTG CTT GCT GAT CCA CAT CTG.
Antibodies directed to peptides within the proteins were obtained commercially (Myosin Va: Sigma, M4812, Myosin Vb: BD Transduction Laboratories, M11220, Myosin VI: Sigma, M5187). Protein was extracted from 1 g of dissected tissue in RIPA buffer containing the appropriate concentration of Roche’s protease inhibitor cocktail tablets. Fifty micrograms of heat denatured cell lysate was loaded onto either a 10% polyacrylamide separating mix and a 5% polyacrylamide stacking gel (when using the semi-dry transfer method) or onto Invitrogen’s 4-12% Bis-Tris gel (when using the wet transfer method).
2.3. Probe preparation and RNA in situ
2.5. Immunohistochemistry
Probes were transcribed from 5034 to 5893 bp of myosin Va (NM_010864), 3491–4331 bp of myosin Vb (NM_201600) and 3345– 4238 bp of myosin VI (NMU49739). Transcription was carried out using
Sections were prepared and dehydrated following normal histology procedure. Heat induced antigen retrieval was carried out in citrate buffer pH 6 when detecting Myosin Vb and VI. Antibodies were used at pre-de-
2.4. Immunoblot detection
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Fig. 6. Myosin VI protein in the developing mouse follicle. (a) Myosin VI localises strongly to the cytoplasm of granulosa cells. (b) Without Myosin VI antibody. (c) Myosin VI localises to oocyte periphery in all very early follicles (arrows). (d) In the growing primary follicle Myosin VI staining in the ooplasm is strong and is also in the granulosa cells (arrow). (e) No Myosin VI is seen in the oocyte of antral follicles. Myosin VI is high at the lateral membranes of granulosa cells and no staining is seen in the lateral or basal membranes of mural granulosa cells adjacent to the theca, however staining is seen in apical membranes in contact with the granulosa cells (arrow). (f) In the large antral follicle Myosin VI is strong in the cytoplasm of mural and cumulus granulosa cells but low in the thecal cells. O, oocyte; GC, granulosa cells; T, thecal cells; AC, antral cavity and OSE, outer surface epithelium. Scale bars: a–b = 100 lm, c–d = 30 lm and e–f = 30 lm.
L. McGurk et al. / Gene Expression Patterns 6 (2006) 900–907 termined concentrations. Sections incubated with the mouse Myosin Vb antibody were first blocked with the Fab fragment of mouse IgG. Fluorescent secondary antibodies were used to detect antibody localisation.
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